High-throughput screening of cellular features using high-resolution light-microscopy; Application for profiling drug effects on cell adhesion

2007 ◽  
Vol 158 (2) ◽  
pp. 233-243 ◽  
Author(s):  
Yael Paran ◽  
Micha Ilan ◽  
Yoel Kashman ◽  
Sofee Goldstein ◽  
Yuvalal Liron ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
High Resolution ◽  
Light Microscopy ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Effects

scholarly journals Development of high-throughput screening assays for profiling snake venom Phospholipase A2 activity after high-resolution chromatographic fractionation

2020 ◽  
Author(s):  
Kristina B.M. Still ◽  
Julien Slagboom ◽  
Sarah Kidwai ◽  
Chunfang Xie ◽  
Bastiaan Eisses ◽  
...  
Keyword(s):  
Fatty Acid ◽  
High Resolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Proinflammatory Mediators ◽  
Ph Indicators ◽  
Snake Venom Phospholipase ◽  
Screening Assays ◽  
Animal Venoms ◽  
Phospholipase A2 Activity

AbstractMany organisms, ranging from plants to mammals, contain phospholipase A2 enzymes (PLA2s), which catalyze the production of lysophospholipids and fatty acid proinflammatory mediators. PLA2s are also common constituents of animal venoms, including bees, scorpions and snakes, and they cause a wide variety of toxic effects including neuro-, myo-, cyto-, and cardio-toxicity, anticoagulation and edema. The aim of this study was to develop a generic method for profiling enzymatically active PLA2s in snake venoms after chromatographic separation. For this, low-volume high-throughput assays for assessment of enzymatic PLA2 activity were evaluated and optimized. Subsequently, the assays were incorporated into a nanofractionation platform that combines high-resolution fractionation of crude venoms by liquid chromatography (LC) with bioassaying in 384-well plate format, and parallel mass spectrometric (MS) detection for toxin identification. The miniaturized assays developed are based on absorbance or fluorescence detection (respectively, using cresol red or fluorescein as pH indicators) to monitor the pH drop associated with free fatty acid formation by enzymatically active PLA2s. The methodology was demonstrated for assessment of PLA2 activity profiles of venoms from the snake species Bothrops asper, Echis carinatus, Echis coloratus, Echis ocellatus, Oxyuranus scutellatus and Daboia russelii russelii.

scholarly journals High-Throughput, High-Resolution Interferometric Light Microscopy of Biological Nanoparticles

ACS Nano ◽  
2020 ◽  
Vol 14 (2) ◽  
pp. 2002-2013 ◽  
Author(s):  
Celalettin Yurdakul ◽  
Oguzhan Avci ◽  
Alex Matlock ◽  
Alexander J. Devaux ◽  
Maritza V. Quintero ◽  
...  
Keyword(s):  
High Resolution ◽  
Light Microscopy ◽  
High Throughput

scholarly journals Multi-parametric characterization of drug effects on cells

F1000Research ◽  
2021 ◽  
Vol 9 ◽  
pp. 1199
Author(s):  
Yael Paran ◽  
Yuvalal Liron ◽  
Sarit Batsir ◽  
Nicola Mabjeesh ◽  
Benjamin Geiger ◽  
...  
Keyword(s):  
Light Microscopy ◽  
High Throughput ◽  
Drug Effects ◽  
Parametric Approach ◽  
Automated Identification ◽  
High Definition ◽  
Complex Functions ◽  
And Control

We present here a novel multi-parametric approach for the characterization of multiple cellular features, using images acquired by high-throughput and high-definition light microscopy. We specifically used this approach for deep and unbiased analysis of the effects of a drug library on five cultured cell lines. The presented method enables the acquisition and analysis of millions of images, of treated and control cells, followed by an automated identification of drugs inducing strong responses, evaluating the median effect concentrations and those cellular properties that are most highly affected by the drug. The tools described here provide standardized quantification of multiple attributes for systems level dissection of complex functions in normal and diseased cells, using multiple perturbations. Such analysis of cells, derived from pathological samples, may help in the diagnosis and follow-up of treatment in patients.

Development of a multiplex high-throughput diagnostic assay for the detection of strawberry crown rot diseases using high-resolution melting analysis

2021 ◽  
Author(s):  
Nan-Yi Wang ◽  
Andre Bueno Gama ◽  
Marcus Vinicius Marin ◽  
Natalia Peres
Keyword(s):  
High Resolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
High Resolution Melting ◽  
High Specificity ◽  
Macrophomina Phaseolina ◽  
Disease Diagnosis ◽  
Management Program ◽  
Crown Rot ◽  
Individual Species

Rapid and accurate disease diagnosis is a prerequisite for an effective disease management program in strawberry production. In Florida, Colletotrichum spp., Phytophthora spp, and Macrophomina phaseolina are the primary microorganisms causing strawberry crown rot. Even though the diseases can be caused by different pathogens, symptoms are indistinguishable and equally devastating. To timely inform strawberry growers of diagnostic results for effective deployment of chemical control practices, we developed a multiplex high-resolution melting (HRM) assay to rapidly and accurately detect the above-mentioned pathogens. The multiplex HRM assays using three pre-designed primer pairs showed high specificity for individual species by generating specific melting peaks without cross-reaction between primers or with other common strawberry pathogens. The amplification limit of the assay was 1 pg of Colletotrichum and Phytophthora and 100 pg of M. phaseolina DNA per 10 μl reaction. However, the presence of different melting peaks was observed in mixed DNA samples and was concentration- and target DNA-dependent. A crude DNA extraction protocol was developed to allow high-throughput screening by minimizing the inhibitory effects. Moreover, we applied the HRM assay to 522 plant samples and found high correlations between conventional pathogen isolation and HRM and between singleplex and multiplex assays. Altogether, this multiplex HRM assay is specific, cost-effective, and reliable for the timely detection of strawberry crown rot pathogens.

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